Synthesis and antimicrobial activity of [3,5-dibromo(dichloro)-1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl]acetic acids and their derivatives

Full text of DOI:10.1023/A:1012799002863

Pharmaceutical Chemistry Journal
Vol. 35, No. 7, 2001
SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF
[3,5-DIBROMO(DICHLORO)-1-HYDROXY-4-OXOCYCLOHEXA-2,5-
DIEN-1-YL]ACETIC ACIDS AND THEIR DERIVATIVES
O. P. Shestak,1 V. L. Novikov,1 E. P. Ivanova,1 and N. M. Gorshkova1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 35, No. 7, pp. 19 – 22, July, 2001.
Original article submitted March 1, 2001.
Amides Id (verongiaquinol) and Ih belonging to a group
of compounds with the general formula I, occurring in nature
in sponges of the Verongidae (Aplysinidae) family, were re-
ported to exhibit a high cytostatic activity in vitro with res-
pect to some tumor cells [1, 2] and a strong inhibiting effect
with resect to Na+, K+, and Ca2+ ATPases [3, 4].
task was to obtain synthetic compounds Ia – Ic and Ie – Ig
and study the spectrum of their antimicrobial activity.
In our experiments, commercial acid III was converted
into 2,6-dibromoacid (IV) with a yield of 97% (against 64%
reported in [6]). The transformation of acid IV into amide
VIII was performed in two stages, IV ® VI and VII ® VIII,
which allowed amide VIII to be obtained with a nearly quan-
titative yield. This procedure was more effective as compa-
red to the previously described direct conversion of acid IV
into amide VIII [7]. In the key stage of ipso-oxidation of the
p-substituted phenols VI – XI, we tested various oxidizing
–
O
O
Br
Br
X
X
COR
·
HO
Ià –Ih
O
agents
including
PhI(OCOCF3)2,
(NH4)2Ce(NO3)6,
II
Ce(SO4)2, RuO4, Tl(ClO4)3, Tl(OAc)3, Pb(OAc)4, and
HNO3. The most effective oxidizer was 100% HNO3 in gla-
cial acetic acid solution.
I: X = Br (a – d), Cl (e – h); R = OH (a, e), OMe (b, f); OEt (c, g),
NH2 (d, h).
O
OH
OH
OH
It was also reported that amides Id and Ih have an inhibi-
ting effect with respect to St. aureus and E. coli [1, 5]. Howe-
ver, the spectrum of antimicrobial activity of this class of
compounds is still insufficiently studied.
Br
Br
X
X
X
X
for Ic, Id
Ià – Ih
AcO
CH2COR
CH2COR
VI – XI
CH2COOH CH2COOH
IV, V
The high biological activity of amides Id and Ih and the
low content of these secondary metabolites in the aforemen-
tioned natural sources, which hinder their thorough pharma-
cological characterization, stimulated the development of va-
rious approaches to the synthesis of these substances. There
are two effective pathways for the synthesis of amides Id and
Ih. In the first scheme, the starting compound is p-hydroxyp-
henylacetic acid (III) and a key stage is the ipso-oxidation of
the corresponding 2,6-dihalogen-substituted p-alkylphenol
VIII or IX [6, 7]. In the second scheme, the starting compo-
und is 2,6-dihalogen-substituted benzoquinone, which is
converted into the target compounds under the action of
low-available lithium reagents at low temperatures [8, 9].
The purpose of this study was to optimize the synthesis
of natural amides Id and Ih proceeding from acid III. Another
XII, XIII
III
X = Br (IV, VI – VIII), Cl (V, IX – XI); R = OMe (VI, IX), OEt (VII, X,
XII), NH2 (VIII, XI, XIII).
In this oxidation stage, somewhat better results were ob-
tained using electrochemical oxidation on a Pt anode in
aqueous HClO4. Indeed, VIII oxidized by HNO3 yields ami-
de Id with a yield of 20%, while the electrochemical oxidati-
on process increases this yield to 31% [10]. The total yields
of p-quinols Ia – Id obtained from acid III were 30, 63, 64,
and 19%, respectively (Table 1). For the chlorine-containing
analogs Ie – Ih obtained by a similar method, the yields were
21, 65, 61, and 27%, respectively. Oxidized with lead triace-
tates, substrates VII and VIII yield, in addition to the target
compounds Ic and Id, their acetates XII and XIII with a total
yield of 22 and 34%, respectively.
1
Pacific-Ocean Institute of Bioorganic Chemistry, Far-East Division, Rus-
sian Academy of Sciences, Vladivostok, Russia.
366
0091-150X/01/3507-0366$25.00 © 2001 Plenum Publishing Corporation

Synthesis of [3,5-Dibromo(dichloro)-1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl]acetic Acids
367
The p-quinols Ia – Ih synthesized as described above are
colorless crystalline substances well soluble in EtOH,
Me2CO, DMSO, DMF, and AcOH, moderately soluble in
aqueous ethanol (2 : 3 v/v), and weakly soluble in water on
heating. The proposed structures were confirmed by the data
of elemental analyses and by the results of IR and 1H NMR
measurements (Table 1).
3,5-Dibromo-4-hydroxyphenylacetic acid (IV). To a
solution of commercial acid III (1.52 g, 0.01 mole) in 50 ml
of glacial acetic acid was added dropwise a solution of 3.30 g
(0.021 mole) of Br2 in 1 ml of the same solvent and the mix-
ture was stirred for 96 h at 15°C. Then the reaction mixture
was diluted with 100 ml of water and the precipitate was se-
parated and crystallized from water to obtain 3.00 g (97%) of
acid IV; m.p., 196 – 197°C (reported m.p., 195 – 196°C [6]);
IR spectrum in KBr (nmax, cm – 1): 1554, 1666, 1710,
EXPERIMENTAL CHEMICAL PART
1
2250 – 3100, 3404; H NMR spectrum in CDCl3 (d, ppm):
The IR spectra were recorded with a Specord 75IR spect-
rophotometer (Germany). The 1H NMR spectra were measu-
red on a Bruker WM-250 (250 MHz) spectrometer (Germa-
ny) using TMS as the internal standard. The melting tempe-
ratures were determined with the aid of a Boethius heating
stage. The data of elemental analyses of the synthesized
compounds agreed with the results of analytical calculations.
The purity of the reaction products was checked by TLC on
Silufol UV-254 plates eluted in a benzene – acetone (7 : 1 to
1 : 1) system. Individual compounds were isolated from the
reaction mixtures by preparative column chromatography on
a SiO2 column (5 – 40 mm) with a sorbent – sorbate ratio of
40 : 1. The yields (recalculated for initial acid III) and some
physicochemical characteristics of the synthesized compo-
unds are presented in Table 1.
3.54 (s, 2H, CH2), 7.43 (s, 2H arom), 8.76 (bs, 1H, OH), 9.93
(bs, 1H, COOH).
4-Hydroxy-3,5-dichlorophenylacetic acid (V). A mix-
ture of commercial acid III (1.52 g, 0.01 mole) and 2.85 g
(0.021 mole) of SO2Cl2 was kept for 1 h until gas evolution
ceases; then 1.76 g (0.013 mole) of SO2Cl2 was added and
the mixture was kept for another 1 h on a boiling water bath
and allowed to stand for 16 h at room temperature. Finally,
the excess SO2Cl2 was completely removed and the residue
was crystallized from water to obtain 1.88 g (85%) of acid V;
m.p., 177 – 179°C (reported m.p., 180 – 183°C [7]); IR spec-
trum in KBr (nmax, cm – 1): 1570, 1660, 1712, 2260 – 3120,
1
3396; H NMR spectrum in CDCl3 (d, ppm): 3.62 (s, 2H,
CH2), 7.33 (s, 2H arom), 8.63 (bs, 1H, OH), 9.81 (bs, 1H,
COOH).
TABLE 1. Yields and Physicochemical Characteristics of [3, 5-Dibromo(dichloro)-1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl]acetic Acids
and Their Derivatives (Ia – Ih, XII, and XIII)
IR spectrum *: n, cm – 1
C = O C(O)R
1700 (CO),
1H NMR spectrum **: d, ppm (J, Hz)
Com- Yield,
Empirical
formula
M.p., °C
pound
%
C = C
1594
C – OH
3474
CH2(s)
2.96
2.6 -H
7.68
Other
Ia
30 195 – 196a C8H6Br2O4
1662
–
2605 – 3110 (OH)
Ib
Ic
63 55 – 57 C9H8Br2O4
1602
1681
1680
1715
1714
3560
3561
2.77
2.77
7.43
7.43
3.80 (s, 3H, COOCH3)
64 124 – 125b C10H10Br2O4 1600
1.33 (t, 3H, J 7.1 Hz, CH2CH3), 4.27
(q, 2H, J 7.1 Hz, CH2CH3)
Id
Ie
19 189 – 191c C8H7Br2NO3 1592
1662
1672
1652 (CO),
3140 (NH2)
3416
3480
2.79
2.92
7.61
7.38
5.99 (bs, 1H, OH), 6.67 (bs, 1H, NH),
7.20 (bs, 1H, NH)
21 177 – 179 C8H6Cl2O4
1594
1609
1704 (CO),
2615 – 3115 (OH)
If
65 73 – 75
61 88 – 90
C9H8Cl2O4
1696
1698
1712
1712
3566
3563
2.80
2.80
7.18
7.21
3.80 (s, 3H, COOCH3)
Ig
C10H10Cl2O4 1607
1.29 (t, 3H, J 7.0 Hz, CH2CH3), 4.24
(q, 2H, J 7.0 Hz, CH2CH3)
Ih
27 160 – 163 C8H7Cl2NO3 1602
1668
1655 (CO),
3422
2.80
7.35
6.03 (bs, 1H, OH), 6.74 (bs, 1H, NH),
7.23 (bs, 1H, NH)
3224 (NH2)
1736
XII
22 86 – 89
C12H12Br2O5 1600
1688
1674
1755
(OAc)
2.89
2.93
7.54
7.78
1.27 (t, 3H, J 7.0 Hz, CH2CH3), 2.09
(s, 3H, OCOCH3), 4.18 (q, 2H, J
7.0 Hz, CH2CH3)
XIII 34 119 – 122 C10H9Br2NO4 1602
1689 (CO),
3190 (NH2)
1754
(OAc)
2.10 (s, 3H, OCOCH3), 6.57 (bs, 1H,
NH), 7.12 (bs, 1H, NH)
*
IR spectra of acids Ia, Ie and amides Id, Ih were recorded using samples pelletized with KBr; other samples were measured as solutions in
CHCl3;
**
1H NMR spectra of acids Ia, Ie and amides Id, Ih were measured in d6-acetone; other compounds were dissolved in CDCl3; a reported m.p.,
195 – 196°C [6]; b reported m.p., 121°C [6], 127 – 127.5°C [8]; c reported m.p., 195 – 196°C [6], 194 – 195°C [8].

368
O. P. Shestak et al.
TABLE 2. Antimicrobial Activity of compounds Ia – Ih, XII, and
XIII
CH2), 4.17 (q, 2H, J 7.2 Hz, CH2CH3), 7.22 (s, 2H arom),
8.35 (bs, 1H, OH).
MIC 100, mg/ml
3,5-Dibromo-4-hydroxyphenylacetamide (VIII).
A
Com-
pound
mixture of 3.38 g (10 mmole) of ester VII, 49 mg (1 mmole)
of NaCN, and 30 ml of a 9 M solution of gaseous NH3 in an-
hydrous MeOH was heated in a stainless-steel hermetic am-
poule at 45°C for 45 h. Then the solvent was distilled off, the
residue was diluted with 50 ml of water, and the product was
extracted with EtOAc (3 ´ 15 ml). The extract was dried
over anhydrous Na2SO4, the solvent was distilled off, and the
residue was chromatographed on a SiO2 column eluted with
a hexane – acetone (1 : 2) mixture to obtain 3.03 g (98%) of
amide VIII; m.p., 182 – 184°C (reported m.p., 181 – 182°C
[7]); IR spectrum in KBr (nmax, cm – 1): 1548, 1650, 3166,
3208, 3401; 1H NMR spectrum in [(CD3)2CO] (d, ppm): 3.45
(s, 2H, CH2), 6.34 (bs, 1H, NH), 6.94 (bs, 1H, NH), 7.50 (s,
2H arom), 8.48 (bs, 1H, OH).
S.
E.
B.
P. aeru-
C.
E. coli
aureus faecium subtilis
ginosa albicans
Ia
Ib
Ic
Id
Ie
If
6.25 > 100 > 100 > 100 > 100 > 100
> 100 > 100 50.0
> 100 > 100 100.0
> 100
> 100
12.5
-²-
-²-
-²-
-²-
-²-
-²-
-²-
-²-
-²-
-²-
-²-
-²-
-²-
-²-
-²-
-²-
-²-
-²-
1.56
12.5
50.0
1.56
> 100 > 100 > 100
> 100 > 100 > 100 100.0
> 100 > 100 > 100 > 100
Ig
Ih
3.12
12.5
3.12
12.5
XII
> 100 > 100 25.0
> 100 > 100 1.56
> 100
50.0
XIII
Tetracy-
cline
0.78
3.12
0.39
3.12
0.39
0.195
3.12
0.78
6.25
3.12
-²-
-²-
-²-
-²-
-²-
-²-
A similar procedure using ester X yields 4-hydro-
xy-3,5-dichlorophenylacetamide (XI); yield, 93%; m.p.,
187 – 190°C; IR spectrum in KBr (nmax, cm – 1): 1564, 1654,
Strepto-
mycin
Oleando-
mycin
1
3192, 3210, 3406; H NMR spectrum in [(CD3)2CO] (d,
0.195 12.5
ppm): 3.46 (s, 2H, CH2), 6.36 (bs, 1H, NH), 6.97 (bs, 1H,
NH), 7.32 (s, 2H arom), 8.80 (bs, 1H, OH).
Amide oxidation by 100% HNO3. To a solution of
618 mg (2.0 mmole) of amide VIII in 3 ml of glacial acetic
acid cooled to 10°C was added in one take 5.3 ml of a mixtu-
re of 100% HNO3 and glacial acetic acid (1 : 9, v/v) cooled
to 10°C, and the solution was stirred at 10 – 15°C for 4.5 h.
Then 30 ml of water was added, the product was extracted
with EtOAc (3 ´ 10 ml), and the extract was dried over an-
hydrous Na2SO4. Finally, the solvent was distilled off and the
residue was chromatographed on a SiO2 column eluted with
a hexane – acetone (3 : 1) mixture to obtain 107 mg (20%) of
2,6-dibromo-4-hydroxy-4-carbamoylmethylcyclohexa-2,5-
dien-1-one (Id) (Table 1).
3,5-Dibromo-4-hydroxyphenylacetic acid methyl es-
ter (VI). A mixture of 620 mg (2.0 mmole) of acid IV, 15 ml
of MeOH, 40 ml of C6H6, and 0.1 ml of concentrated H2SO4
was kept boiling in a setup equipped with a Dean – Stark at-
tachment. Then the mixture was washed with of water
(3 ´ 10 ml) and dried over anhydrous Na2SO4. Finally, the
solvent is distilled off to obtain 642 mg (99%) of ester VI;
m.p., 106 – 107°C (hexane); IR spectrum in CHCl3 (nmax
,
1
cm – 1): 1564, 1737, 3504; H NMR spectrum in CDCl3 (d,
ppm): 3.53 (s, 2H, CH2), 3.73 (s, 3H, COOMe), 7.40 (s,
2H arom), 8.49 (bs, 1H, OH).
Analogous procedures were used to obtain 2,6-dibro-
mo-4-hydroxy-4-hydroxycarbonylmethylcyclohexa-2,5-di-
en-1-one (1a) from acid IV (yield, 31%); 2,6-dibromo-4-hyd-
roxy-4-methoxycarbonylmethylcyclohexa-2,5-dien-1-one
(1b) from ester VI (yield, 67%); 2,6-dibromo-4-hydro-
xy-4-ethoxycarbonylmethylcyclohexa-2,5-dien-1-one (1c)
from ester VII (yield, 68%); 2,6-dichloro-4-hydroxy-4-hyd-
roxycarbonylmethylcyclohexa-2,5-dien-1-one (1d) from acid
V (yield, 25%); 2,6-dichloro-4-hydroxy-4-methoxycarbonyl-
methylcyclohexa-2,5-dien-1-one (1f) from ester IX (yield,
78%); 2,6-dichloro-4-hydroxy-4-ethoxycarbonylmethylcyc-
lohexa-2,5-dien-1-one (1g) from ester X (yield, 74%); and
2,6-dichloro-4-hydroxy-4-carbamoylmethylcyclohe-
A similar procedure using acid V was used for the synt-
hesis of 4-hydroxy-3,5-dichlorophenylacetic acid methyl es-
ter (IX); yield, 98%; m.p., 83 – 85°C (hexane); IR spectrum
1
in CHCl3 (nmax, cm – 1): 1578, 1736, 3524; H NMR spect-
rum in CDCl3 (d, ppm): 3.52 (s, 2H, CH2), 3.72 (s, 3H, COO-
Me), 7.20 (s, 2H arom), 8.36 (bs, 1H, OH).
A similar procedure using acid IV and EtOH instead of
MeOH gives 3,5-dibromo-4-hydroxyphenylacetic acid ethyl
ester (VII); yield, 98%); m.p., 105 – 106°C (hexane), repor-
ted m.p., 105°C [6]; IR spectrum in CHCl3 (nmax, cm – 1):
1
1566, 1730, 3506; H NMR spectrum in CDCl3 (d, ppm):
1.26 (t, 3H, J 7.2 Hz, CH2CH3), 3.52 (s, 2H, CH2), 4.17 (q,
2H, J 7.2 Hz, CH2CH3), 7.40 (s, 2H arom), 8.48 (bs, 1H,
OH).
A similar procedure using acid V and EtOH yields
4-hydroxy-3,5-dichlorophenylacetic acid ethyl ester (X);
yield, 97%; m.p., 75 – 76°C (hexane); IR spectrum in CHCl3
(nmax, cm – 1): 1587, 1728, 3527; 1H NMR spectrum in
CDCl3 (d, ppm): 1.27 (t, 3H, J 7.2 Hz, CH2CH3), 3.51 (s, 2H,
xa-2,5-dien-1-one (1h) from amide XI (yield, 35%).
Amide oxidation by Pb(OAc)4. Synthesis of compo-
unds XII and XIII. A mixture of 224 mg (0.725 mmole) of
amide VIII, 800 mg (1.8 mmole) of freshly prepared
Pb(OAc)4, and 2 ml of glacial acetic acid was stirred at room
temperature for 5 h. Then 15 ml of water was added and the
product was extracted with EtOAc (3 ´ 5 ml). The extract

Synthesis of [3,5-Dibromo(dichloro)-1-hydroxy-4-oxocyclohexa-2,5-dien-1-yl]acetic Acids
369
was dried over anhydrous Na2SO4, the solvent was distilled
off, and the residue was chromatographed on a SiO2 column
eluted with a hexane – acetone (3 : 5) mixture to obtain
96 mg (36%) of 4-acetoxy-2,6-dibromo-4-carbamoylmethyl-
cyclohexa-2,5-dien-1-one (XIII) (Table 1). Then the column
was eluted with a hexane – acetone (1 : 2) mixture to obtain
20 mg (9%) of amide Id analogous to that synthesized as des-
cribed above.
A similar procedure using ester VII gives 4-aceto-
xy-2,6-dibromo-4-ethoxycarbonylmethylcyclohexa-2,5-di-
en-1-one (XII) with a yield of 23% and p-quinol Ic (yield,
13%) (Table 1)
ficantly inhibited only by amides Id and Ih, but even these
compounds could only moderately inhibit the growth of E.
faecium.
It should be noted that acylation of the OH group in posi-
tion 4 of amide Id (compound VIII) leads to a sharp drop in
activity with respect to St. aureus and E. faecium, but not to
B. subtilis. Moreover, acylation of this OH group in ester Ic
even increases the activity with respect to B. subtilis (cf. data
for compounds Ic and XII). As for the Gram-negative species
of E. coli, only amides Id and Ih show a moderate activity
with respect to these bacteria. Other Gram-negative species
such as P. aeruginosa, as well as C. albicans yeasts, are in-
sensitive to the synthesized compounds.
The results of our investigation indicate that some deri-
vatives of (3,5-dihalogen-substituted 1-hydroxy-4-oxocyclo-
hexa-2,5-dien-1-yl)acetic acids, such as amides Id and Ih, are
of interest as potential antimicrobial agents.
EXPERIMENTAL BIOLOGICAL PART
The antimicrobial activity of compounds Ia – Ih, XII,
and XIII was studied with respect to test cultures of
Gram-positive (Staphylococcus aureus IFO 14462, Entero-
coccus faecium CIP 104105, Bacillus subtilis ATCC 6051 T)
and Gram-negative (Escherichia coli IFO 15034, Pseudomo-
nas aeruginosa KMM 433) bacteria and Candida albicans
KMM 455 yeast cultures obtained from collections of the
Pasteur Institute (CIP, France), Fermentation Institute (IFO,
Japan), American Typical Culture Collection (ATCC, USA),
and Sea Microorganism Culture Collection (Pacific-Ocean
Institute of Bioorganic Chemistry, Vladivostok, Russia). The
antimicrobial tests were performed as described previously
[11] using a standard method of diffusion into agar in the me-
dia featuring optimum growth of the test microorganism cul-
tures. The content of microbial cells per ml in the test sus-
pensions was (1.0 – 2.0) ´ 108 (OD660 = 0.13 – 0.20). Dia-
meters of the zones of test culture growth inhibition were
determined after incubation for 20 – 24 h in a thermostat at
37°C. The activity of compounds was evaluated as the mini-
mum inhibiting concentration (MIC, mg/ml) completely sup-
pressing the growth of test cultures. The reference drugs
were tetracycline, streptomycin, and oleandomycin.
ACKNOWLEDGMENTS
This study was partly supported by the Russian Founda-
tion for Basic Research, project No. 00-15-97397.
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are most active with respect to Gram-positive bacterial speci-
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type of radical R in the two-carbon side chain (Table 2). Only
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